Insulin is a kind of peptide hormone which is critical for glucose homeostasis. The mature Insulin peptide is derived from Proinsulin, which includes the Insulin A and B chains connected by a peptide fragment (C-peptide). Proinsulin is processed within the endoplasmic reticulum of pancreatic beta cells into equimolar ratios of mature Insulin and C-peptide. Mouse C-peptide 1 is a single chain peptide composed of 29 amino acids, while C-peptide 2 is composed of 31 residues. C-peptide is secreted together with insulin.

 

The role of C-peptide has been considered to keep the best configuration to form three disulfide bonds, and has no biological activity, however, recent studies indicated that C-peptide can bind, probably, a G-protein coupling specific receptor present on the surface of endothelial cells, kidney microtubule cells and fibroblasts, resulting in activation of calcium-dependent intracellular signaling, activation of Na+-K+-ATPase, and enhancement of NO synthesis. Administration of C-peptide to DM1 patients enhances blood circulation in the skeletal muscle and skin, and also minimizes kidney glomerular hyperfiltration, decreasing albumin excretion into urine, and also improves nervous function, indicating that C-peptide should be given together with insulin to DM1 patients. Important region to bind receptor has been reported to be C-terminal penta peptide.

This assay is a two-site ELISA. The micro-plate is pre-coated with a monoclonal antibody against C-peptide. Standards and samples are added into the wells and co-incubated with a biotin labeled monoclonal antibody. After wash step to remove any unbound reagents, streptavidin-HRP conjugate (STP-HRP) is added. After the last wash step, TMB substrate is added and colour develops in proportion to the amount of C-peptide bound initially. The assay is stopped and the optical density of the wells determined using a micro-plate reader. Since the increases in absorbance are directly proportional to the amount of captured C-peptide, the unknown sample concentration can be interpolated from a reference curve included in each assay.

A. Typical representation of standard curve

The following standard curve is provided for demonstration only. A standard curve should be generated for each assay.

C-peptide (ng/mL)	Absorbance (450 nm)	Blanked Absorbance
0	0.133	0
0.3	0.151	0.018
0.6	0.203	0.07
1.2	0.338	0.205
2.4	0.783	0.65
6	2.479	2.346

 

B.Sensitivity

The lowest level of Mouse C-peptide that can be detected by this assay is 0.3 ng/mL.

 

C. Precision

Intra-assay Precision (Precision within an assay) C.V. <10%.

Inter-assay Precision (Precision between assays) C.V. <10%.